The present invention concerns prolonged enhanced chemiluminescence. More particularly, the present invention relates to stabilization of the enzyme in enhanced chemiluminescence reactions by the use of nitrogen-containing compounds.
Luminescence is defined as the emission of light without heat. In luminescence, energy is specifically channeled to a molecule so that a specific light-emitting state is produced without greatly increasing the temperature of the molecule. The color is determined by the character of the light-emitting state involved, and does not change when the energy or method to produce it is changed.
Chemiluminescence is defined as luminescence wherein a chemical reaction supplies the energy responsible for the emission of light (ultraviolet, visible, or infrared) in excess of that of a blackbody (thermal radiation) at the same temperature and within the same spectral range. Chemiluminescence thus involves the direct conversion of chemical energy to light energy. Below 500.degree. C., the emission of any light during a chemical reaction involves chemiluminescence. The blue inner cone of a bunsen burner or the Coleman gas lamp are examples.
Many chemical reactions generate energy. Usually this exothermicity appears as heat, that is, translational, rotational, and vibrational energy of the product molecules; whereas, for a visible chemiluminescence to occur, one of the reaction products must be generated in an excited electronic state (designated below by an asterisk) from which it can undergo deactivation by emission of a photon. Hence a chemiluminescent reaction, as shown in reactions (a) and (b) below, can be regarded as the reverse of a photochemical reaction. EQU A+B.fwdarw.C*+D (a) EQU C*.fwdarw.C+h (b)
The energy of the light quantum h.nu. (where h is Planck's constant, and .nu. is the light frequency) depends on the separation between the ground and the first excited electronic state of C; and the spectrum of the chemiluminescence usually matches the fluorescence spectrum of the emitter. Occasionally, the reaction involves an additional step, the transfer of electronic energy from C* to another molecule, not necessarily otherwise involved in the reaction. Sometimes no discrete excited state can be specified, in which case the chemiluminescence spectrum is a structureless continuum associated with the formation of a molecule, as in the so-called air afterglow: NO+O.fwdarw.NO.sub.2 +h.nu.(green light).
The efficiency of a chemiluminescence is expressed as its quantum yield .phi., that is, the number of photons emitted per reacted molecule. Many reactions have quantum yields much lower (10.sup.-8 h.nu. per molecules) than the maximum of unity, Einsteins of visible light (1 einstein=Nh.nu., where N is Avogadro's number), with wavelengths from 400 to 700 nm, correspond to energies of about 70 to 40 kcal per mole (300 to 170 kilojoules per mole). Thus only very exothermic, or "exergonic," chemical processes can be expected to be chemiluminescent. Partly for this reason, most familiar examples of chemiluminescence involve oxygen and oxidation processes; the most efficient examples of these are the enzyme-mediated bioluminescences. The glow of phosphorus in air is a historically important case, although the mechanism of this complex reaction is not fully understood. The oxidation of many organic substances, such as aldehydes or alcohols, by oxygen, hydrogen peroxide, ozone, and so on, is chemiluminescent. The reaction of heated ether vapor with air results in a bluish "cold" flame, for example. The efficiency of some chemiluminescences in solution, such as the oxidation of luminol (I) (see formula below) and, especially, the reaction of some oxalate esters (II) (see formula below) with hydrogen peroxide, can be very high (.phi.=30%). ##STR1##
It is believed that the requirements for chemiluminescence are not only sufficient exothermicity and the presence of a suitable emitter, but also that the chemical process be very fast and involve few geometrical changes, in order to minimize energy dissipation through vibrations. For example, the transfer of one electron from a powerful oxidant to a reductant (often two radical ions of opposite charge generated electrochemically) is a type of process which can result, in some cases, in very effective generation of electronic excitation. An example, with 9,10-diphenylanthracene (DPA), is shown in reaction (c). EQU DPA.sup.- +DPA.sup.+ .fwdarw.DPA.sup.* +DPA (c)
The same is true of the decomposition of four-membered cyclic peroxides (III) into carbonyl products, shown in reaction (d), which may be the prototype of many chemiluminescences. ##STR2##
A special type of chemiluminescence is bioluminescence.
Bioluminescence is defined as the emission of light by living organisims, due to an energy-yielding chemical reaction in which a specific biochemical substance, called luciferin, undergoes oxidation, catalyzed by a specific enzyme called luciferase.
There are many specific luciferins and luciferases which are chemically different, each involved in some different living luminescent organism. The flash of the firefly, the brilliant "phosphorescence" or "burning" of the ocean, or the eerie glow of mushrooms deep in the forest at night are but a few examples of these different bioluminescent organisms.
Since bioluminescence is a type of chemiluminescence, it is not necessary to have a live organism to obtain light emission. The simple preservation of the chemicals involved will suffice. This can be done in some cases by rapidly drying the organism under mild conditions.
Dried firefly tails (lanterns) emit light when ground up with water. This light emission dies away within a few minutes, but can be restored by the addition of adenosinetriphosphate (ATP), a key coenzyme in the energy metabolism of cells. In this case, ATP reacts with the luciferin of fireflies to give the luciferyl adenylate intermediate and pyrophosphate (PP).
Using lantern extracts from hundreds of thousands of fireflies, scientists at Johns Hopkins University determined the chemical structure of firefly luciferin to be C.sub.13 H.sub.12 N.sub.2 O.sub.3 S.sub.2. It can now be synthesized. The reaction of luciferyl adenylate with oxygen is postulated to give a four-membered-ring alpha-peroxylactone intermediate and to release adenosinemonophosphate (AMP). This breaks down in the energy-yielding step to give carbon dioxide and a light-emitting excited molecule. This loses its energy as a photon (h.nu.), in the yellow region of the spectrum in this case.
Firefly luciferin and luciferase from preserved light organs are used in a very sensitive biochemical test to detect AIP.
A postulated pathway for firefly luciferin is as follows: ##STR3##
The luminescence of the firefly occurs as a brief flash, coming from the inside of photogenic cells in the lantern, under the control of the nervous system. Quite a different situation occurs in the small marine crustacean Cypidina, which is found in the waters off the coast of Japan. It synthesizes its luciferin and luciferase in separate glands. To emit light, it simply squirts luciferin and luciferase into the water, where the reaction occurs, separate from the animal. The light may function to divert or trick predators.
The chemistry of Cypridina luciferin has been determined by a group of chemists in Japan. C.sub.22 H.sub.27 ON.sub.7 is postulated to react directly with oxygen at the position indicated by the arrow (below), forming a type of alphaperoxylactone similar to the firefly molecule. In the final step, carbon dioxide is also released, along with the excited molecule, which in this case emits in the blue.
A postulated pathway for Cypridina luciferin is as follows: ##STR4##
Like fireflies, dried Cypridina emit light when ground up with cool water; the preserved luciferin and luciferase are released from the glands as they are crushed. The light gradually fades as the luciferin is oxidized, but the addition of more luciferin restores light in the exhausted extract. Luciferin can be obtained either synthetically, or in the natural form by grinding up dried Cypridina in hot water. The heat destroys the luciferase, which is a protein, but leaves the luciferin active. When cooled and mixed with the exhausted extract, luminescence is observed. This is the basis for the classical luciferin-luciferase test.
Luminescent bacterial emit a continuous blue-green light. Such bacteria can be isolated directly from sea water or from the surface of a dead fish and will grow rapidly on any medium containing 3% salt (equivalent to sea water) and some fish or meat extract.
A postulated pathway for bacteria luciferin is as follows: ##STR5##
Chemiluminescent detection is one of the most sensitive ways of detecting an analyte. The process, although sensitive, suffers from several disadvantages. In most cases the chemiluminescent reaction mediated emission of light has a very short lifetime, i.e., light emission is very quick, so that a sophisticated device has to be developed to monitor the extent of light emission and also to determine the extent of the presence of an analyte. It is also difficult to couple the interacting systems to the analyte without destroying or changing the property of the interacting partners.
Recently, it has been demonstrated that if a substance, for example, an iodophenol or a benzothiazole derivative is present during the chemiluminescent emission mediated by horseradish peroxidase, the reaction rate is retarded and simultaneously the quantum yield of the light emission is enhanced (European Patent Application No. 0 116 454; European Patent Application No. 0 103 784; UK Patent Application No. 820 62 63; Gary H. G. Thorpe, Robert Haggart, Larry J. Kricka and Thomas P. Whitehead, "Enhanced Luminescent Enzyme Immunoassays For Rubella Antibody, Immunoglobulin And Digoxin", Biochemical and Biophysical Research Communications, Vol. 119, No. 2, pp. 481-487, Mar. 15, 1984; Thomas P. Whitehead, Gary H. G. Thorpe, Timothy J. N. Carter, Carol Groucutt and Larry J. Kricka, "Enhanced Luminescence Procedure For Sensitive Determination Of Peroxidase-labelled Conjugates In Immunoassay", Nature, Vol. 305, pp. 158-159, Sept. 8, 1983.
Irwin Fridovich, "The Stimulation Of Horseradish Peroxidase By Nitrogenous Ligands", The Journal of Biological Chemistry, Vol. 238, No. 12, December 1963, pp. 3921-3927, describes the stabilization of peroxidase in solution with nitrogenous ligands.
It has been demonstrated heretofore that a chemiluminescent reaction occurs where the emission is due to an iron initiated activation of bleomycin. The self-inactivation reaction is affected by the presence of DNA.
In Photochemistry Photobiology, Vol. 40, pg 823-830, (1984), it was described that photoemission is quenched by target molecules such as DNA and that the presence of DNA does not prevent the iron-initiated activation of bleomycin, by the so-called self-inactivation reaction associated with chemiluminescence. The article went on to state that these findings seem to suggest that an electronically excited intermediate of bleomycin can alter bio-molecules though, in that case, the nature of the excited state was not precise.
Swedish patent application No. 8200479 describes chemiluminescent detection of nucleic acid hybrids.
European patent application No. 0 070 687 concerns a light-emitting polynucleotide hybridization diagnostic method.
Heretofore chemiluminescene reactions proceeded too quickly and thus resulted in light of only a short duration. The use of enhancers have somewhat extended and amplified the light from chemiluminescence reactions, however, the duration and intensity of the emitted light is still in many instances inadequate.
Immunoassay is one of the most widely used analytical techniques in the clinical laboratory. At present the majority of immunoassays employ a radioactive isotope, especially iodine-125, as a label. However, radioactive isotopes have a number of major disadvantages. First, the method of labelling involves the use of highly radioactive and hence potentially hazardous reagents. Second, the shelf life of the radio-actively labelled substance is often relatively short not only because by its very nature the radioactive isotope is continuously decaying but also because radioactively labelled proteins are often unstable. Third, it is often difficult to label proteins sufficiently to provide a sensitively and rapidly detectable reagent. Fourth, the disposal of radioactively labelled substances is inconvenient.
These disadvantages have stimulated a search for viable alternatives to the radio label. To be suitable as a label a substance should meet at least the following three requirements:
a. it should be detectable both rapidly and in very small quantities when attached to a ligand such as an antigen or an antibody; PA1 b. it should be possible to attach it, without affecting its determination, to a ligand such as an antigen or an antibody; and PA1 c. once attached, it should not significantly alter the properties of the ligand. PA1 a. Organoluminescent or organoluminometric immunoassays wherein chemiluminescent or bioluminescent compounds which participate directly in luminescent reactions (i.e., which are converted to an excited state and then return to a non-excited state with the emission of a photon) have been used to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. Examples of suitable compounds include luminol and isoluminol; PA1 b. Luminescent catalyst or cofactor immunoassays wherein catalysts or cofactors of luminescent reactions have been used as labels. An example of a suitable catalyst is the enzyme peroxidase; and PA1 c. Enzyme linked immunoassays wherein luminescent reactions have been used to determine the products formed by the action of enzyme labels on suitable substrates. An example of this type of immunoassay is the determination of antibody linked glucose oxidase by reacting the enzyme/antibody reagent with glucose to form hydrogen peroxide and then measuring the amount of hydrogen peroxide produced by adding luminol under controlled conditions to initiate a luminescent reaction.
Some of the most promising alternative labels are either substances which can themselves take part in a reaction resulting in the emission of luminescent light or substances which, on suitable treatment, produce compounds capable of taking part in a luminescent reaction. Heretofore, the use of luminescence in immunoassays has suffered since the measurement of luminescence is a rapid process and may be completed in a matter of seconds rather, than the several minutes generally required for the measurement of radioactivity.
Luminescence has been employed in three major luminescent or luminometric immunoassay systems:
The sensitivity of the above assays is determined in part by the lower limit for detection of the label or the product of the label. In the case of luminescent or luminometric assays the sensitivity of the system will depend partially on the light emitted in the luminescent reaction per unit of labelled material.